The present invention relates to soluble plastid processing enzymes. In particular, the present invention relates to a chloroplast processing enzyme (CPE), that is an endoprotease with unique cleavage capabilities, a cDNA encoding the enzyme, and the use of the enzyme in preparing biologically active (native) polypeptides for various applications.
Enzymes (e.g. proteases) are valuable tools for use in the production of peptides and polypeptides. However, many of the known proteases cleave at so many sites in a molecule that the resultant products are not biologically active, and/or the enzymes cleave at common sites so that there is no cleavage product specificity. Specialized enzymes such as those that convert biologically inactive precursor molecules to active molecules by retaining specific portions of the molecule, would be assets to protein production, because their products could be predictable, and biologically active. Although some of these processing enzymes are known, difficulties in producing them in quantities suitable for commercial use, seriously limit their use. Some difficulties arise from problems in isolating and purifying the enzymes, other difficulties stem from the source material--many are obtained from animals which are a less desirable source than plants.
In plants, certain systems are advantageous as a source of enzymes because they produce products of general importance. For example, the chloroplast serves as the site for many biosynthetic pathways such as fatty acid synthesis, terpene synthesis, aromatic and branched amino acid synthesis, starch accumulation, nitrogen and sulfur reduction, photosynthesis, ATP generation, and carbon-dioxide fixation. Therefore, methods and compositions in the chloroplast are broadly applicable to non-chloroplast applications. For example, terpenes are compounds that were originally isolated from the oil of turpentine in the early days of organic chemistry. Terpene derivatives, including alcohols, aldehydes, and esters are referred to as terpenoids. Terpenoids are a category of chemicals responsible for the aromatic characteristics of fragrances. The terpenoid molecular structure is based on five-carbon units. Different chemical arrangements of the basic five-carbon units produce terpenoid compounds with different scents, such as lemongrass, lavender, menthol, jasmine, violet, and camphor. Methods and compositions from chloroplasts may be applied to the perfume industry by producing terpenoids in a manner suitable for commercial use.
Chloroplast biogenesis depends upon the import of many diverse proteins, which are synthesized in the cytoplasm as pre-proteins with N-terminal transit peptides. The transit peptide mediates pre-protein recognition by receptors on the chloroplast envelope (Schnell et al., 1994; Hirsch (et al., 1994). Upon membrane translocation into the stroma, the transit peptide is proteolytically removed, yielding a mature protein (Abad et al., 1989; Robinson et al., 1984). It has been suggested that a general stromal processing peptidase (SPP) located within the chloroplast, exhibiting the properties of a metalloprotease, cleaves the transit peptides from the diverse group of pre-proteins that are imported into the chloroplast (Abad et al., 1989; Robinson et al., 1984).
Proteins targeted to the thylakoid lumen have a bipartite transit peptide that is cleaved first by SPP, then by a thylakoid protease (Bassham et al., 1991; Kirwin et al., 1988; Konishi et al., 1993). SPP thus plays a key role in the maturation process of proteins targeted to the chloroplast. Identification and characterization of genes encoding processing proteases (peptidases) may facilitate the development of recombinant expression systems that require expression of pre-proteins and subsequent processing to remove transit peptides or other segments of the sequence that must be removed in order to yield mature proteins.